BRENDA - Enzyme Database
show all sequences of 1.1.2.B3

Quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: the unusual disulfide ring formed by adjacent cysteine residues is essential for efficient electron transfer to cytochrome c550

Mennenga, B.; Kay, C.W.; Goerisch, H.; Arch. Microbiol. 191, 361-367 (2009)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
Amine
the enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 does not depend on an activating amine which is essential for wild type activity under these condition
Pseudomonas aeruginosa
ethylamine
5 mM, wild-type enzyme shows strict dependence on an activator
Pseudomonas aeruginosa
Cloned(Commentary)
Cloned (Commentary)
Organism
-
Pseudomonas aeruginosa
Engineering
Protein Variants
Commentary
Organism
C105A/C106A
all pyrroloquinoline quinone-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure is generated by replacing Cys105 and Cys106 with Ala. Heterologously expressed C105A/C106A apoenzyme is successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor pyrroloquinoline quinone (PQQ) in the presence of Ca2+. The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 does not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme shows increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol is unaltered. For all substrates tested the specific activity of the mutant enzyme in the artificial dye test is higher than that found for wild type enzyme. In the ferricyanide test with the natural electron acceptor cytochrome c550 the activity of mutant Cys105Ala/Cys106Ala is 15fold lower than that of wild type enzyme
Pseudomonas aeruginosa
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.012
-
ethanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
0.062
-
1-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
0.43
-
1-butanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
1.4
-
2-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
1.5
-
2-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
3.8
-
1-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
12
-
ethanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
12
-
1-butanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
21
-
1,3-Propanediol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
266
-
1,3-Propanediol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
alcohol + cytochrome c550
Pseudomonas aeruginosa
the unusual disulfide ring between the adjacent cysteine residues C105 and C106 is essential for efficient electron transfer at pH 7 from quinoprotein ethanol dehydrogenase to its natural electron acceptor cytochrome c550
aldehyde + reduced cytochrome c550
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Pseudomonas aeruginosa
Q9Z4J7
ATCC17933
-
Specific Activity [micromol/min/mg]
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
11
-
substrate: 2-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
11
-
substrate: ethanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
15
-
substrate: 1-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
20
-
substrate: ethanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
40
-
substrate: 1,3-propanediol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
42
-
substrate: 1-butanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
52
-
substrate: 1-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
94
-
substrate: 2-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
224
-
substrate: 1,3-propanediol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
461
-
substrate: 1-butanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
1,3-propanediol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
? + reduced acceptor
-
-
-
?
1-butanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
butanal + reduced acceptor
-
-
-
?
1-propanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
propanal + reduced acceptor
-
-
-
?
2-propanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
2-propanone + reduced acceptor
-
-
-
?
alcohol + cytochrome c550
the unusual disulfide ring between the adjacent cysteine residues C105 and C106 is essential for efficient electron transfer at pH 7 from quinoprotein ethanol dehydrogenase to its natural electron acceptor cytochrome c550
690646
Pseudomonas aeruginosa
aldehyde + reduced cytochrome c550
-
-
-
?
ethanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
acetaldehyde + reduced acceptor
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
QEDH
-
Pseudomonas aeruginosa
quinoprotein ethanol dehydrogenase
-
Pseudomonas aeruginosa
Cofactor
Cofactor
Commentary
Organism
Structure
pyrroloquinoline quinone
-
Pseudomonas aeruginosa
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
Amine
the enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 does not depend on an activating amine which is essential for wild type activity under these condition
Pseudomonas aeruginosa
ethylamine
5 mM, wild-type enzyme shows strict dependence on an activator
Pseudomonas aeruginosa
Cloned(Commentary) (protein specific)
Commentary
Organism
-
Pseudomonas aeruginosa
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
pyrroloquinoline quinone
-
Pseudomonas aeruginosa
Engineering (protein specific)
Protein Variants
Commentary
Organism
C105A/C106A
all pyrroloquinoline quinone-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure is generated by replacing Cys105 and Cys106 with Ala. Heterologously expressed C105A/C106A apoenzyme is successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor pyrroloquinoline quinone (PQQ) in the presence of Ca2+. The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 does not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme shows increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol is unaltered. For all substrates tested the specific activity of the mutant enzyme in the artificial dye test is higher than that found for wild type enzyme. In the ferricyanide test with the natural electron acceptor cytochrome c550 the activity of mutant Cys105Ala/Cys106Ala is 15fold lower than that of wild type enzyme
Pseudomonas aeruginosa
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.012
-
ethanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
0.062
-
1-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
0.43
-
1-butanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
1.4
-
2-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
1.5
-
2-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
3.8
-
1-propanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
12
-
ethanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
12
-
1-butanol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
21
-
1,3-Propanediol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
266
-
1,3-Propanediol
pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
alcohol + cytochrome c550
Pseudomonas aeruginosa
the unusual disulfide ring between the adjacent cysteine residues C105 and C106 is essential for efficient electron transfer at pH 7 from quinoprotein ethanol dehydrogenase to its natural electron acceptor cytochrome c550
aldehyde + reduced cytochrome c550
-
-
?
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
11
-
substrate: 2-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
11
-
substrate: ethanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
15
-
substrate: 1-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
20
-
substrate: ethanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
40
-
substrate: 1,3-propanediol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
42
-
substrate: 1-butanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, wild-type enzyme
Pseudomonas aeruginosa
52
-
substrate: 1-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
94
-
substrate: 2-propanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
224
-
substrate: 1,3-propanediol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
461
-
substrate: 1-butanol, pH 9 in the artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol in the presence of 5 mM ethylamine, mutant enzyme C105A/C106A
Pseudomonas aeruginosa
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
1,3-propanediol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
? + reduced acceptor
-
-
-
?
1-butanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
butanal + reduced acceptor
-
-
-
?
1-propanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
propanal + reduced acceptor
-
-
-
?
2-propanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
2-propanone + reduced acceptor
-
-
-
?
alcohol + cytochrome c550
the unusual disulfide ring between the adjacent cysteine residues C105 and C106 is essential for efficient electron transfer at pH 7 from quinoprotein ethanol dehydrogenase to its natural electron acceptor cytochrome c550
690646
Pseudomonas aeruginosa
aldehyde + reduced cytochrome c550
-
-
-
?
ethanol + acceptor
artificial dye test at fixed concentrations of N-methylphenazonium methyl sulfate and 2,6-dichlorophenol indophenol
690646
Pseudomonas aeruginosa
acetaldehyde + reduced acceptor
-
-
-
?
Other publictions for EC 1.1.2.B3
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [░C]
Temperature Range [░C]
Temperature Stability [░C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [░C] (protein specific)
Temperature Range [░C] (protein specific)
Temperature Stability [░C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
690646
Mennenga
Quinoprotein ethanol dehydroge ...
Pseudomonas aeruginosa
Arch. Microbiol.
191
361-367
2009
2
-
1
-
1
-
-
10
-
-
-
1
-
1
-
-
-
-
-
-
10
-
6
-
2
-
-
-
-
-
-
-
1
-
-
-
2
-
1
1
-
1
-
-
-
-
10
-
-
-
1
-
-
-
-
-
-
10
-
6
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
686425
Arias
Genetic analyses and molecular ...
Pseudomonas putida
Environ. Microbiol.
10
413-432
2008
-
-
2
-
2
-
-
-
1
-
1
2
-
2
-
-
-
-
-
-
-
-
2
2
7
-
-
-
-
-
-
-
3
-
-
-
-
-
2
3
-
2
-
-
-
-
-
1
-
1
2
-
-
-
-
-
-
-
-
2
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
688852
Schobert
A soluble two-component regula ...
Pseudomonas aeruginosa
Microbiology
147
363-372
2001
-
-
1
-
1
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
2
-
4
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-